Constructions Of Novel DNA Biosensors Based On Fluorescence Quenching With Graphene Oxide

Quantitative detection of biomolecules such as DNA and proteins that carry the important information of life processes, is of great importance. DNA biosensor has received tremendous attention in the assay of biomolecules because of its high sensitivity, low cost, easy operation, rapid analysis, etc. Furthermore, the presence of functional nucleic acid such as aptamer, nucleic acid enzyme, aptazyme, further broadens the detection range of DNA biosensor, and improves the detection sensitivity and selectivity.Since its first discovery in2004, graphene has attracted great attention owing to its unique optical, electrical, mechanical, thermal properties and2D nano characteristics. As one derivative of graphene, graphene oxide (GO) possesses good water-solubility, fluorescence properties, superior fluorescence quenching ability, etc. By combining the above features of GO with the properties of DNA, GO has been widely exploited in DNA biosensor. However, there are still some issues that have to be considered in GO-based DNA biosensor:(1) Although the introduction of aptamers increases the detection range of DNA biosensor, not all the molecules to be targeted have aptamers, therefore, how can we construct GO-based platform for the detection of the molecules that have no nucleic acid recognition elements?(2) The study on the effect of GO on the property of DNA adsorbed on its surface is not detailed and complete.(3) Besides being used as a quencher, is GO usable for other sensing applications with its non-covalent interaction with DNA and unique quenching ability? Inspired by these concerns, we herein employed the excellent fluorescence quenching ability of GO to design some novel DNA biosensors for the sensitive detection of small molecule, protein and DNA. The main research work of this dissertation is summarized as follows:(1) We successfully synthesized GO and characterized its morphology and chemical structure. Then, based on the resistance of metal-mediated molecular-beacon (MB) toward nuclease digestion, combined with the binding between biothiols and metal and the remarkable difference in the affinity of GO with MB and the mononucleotides, we propose a novel fluorescence "turn-on" sensor for the assay of biothiols that have no nucleic acid recognition element. The structure of thymine-Hg2+-thymine (T-Hg2+-T) mediated MB was chosen as the model and used for the detection of glutathione with a detection limit of1.53nM. Furthermore, cytosine-Ag+-cytosine (C-Ag+-C) mediated MB was also constructed for the assay of cysteine to demonstrate the versatility of the design, and the detection limit is12.5nM. Furthermore, satisfactory results were also obtained for the determination of biothiols in1%human serum. This method is low-cost, sensitive, and selective.(2) Based on the specific deamination reaction of adenosine deaminase (ADA) to adenosine (AD), combined with the remarkable difference in the affinity of GO with aptamer and aptamer/target complex, we present a novel fluorescent aptasensor for the detection of ADA activity. This GO-based design which needs only one labeled single-stranded DNA, is not only cost-effective, but also simple, fast and sensitive. The detection limit is1.54nM, which is more than one order of magnitude lower than the electrochemical measurements. Furthermore, satisfactory results were also obtained for the determination of ADA activity in5%human serum.(3) In the previous chapter, we constructed a detection method for the activity of adenosine deaminase based on the different binding ability of aptamer to its substrate adenosine (AD) and the product. However, if the product can also bind with the aptamer, this strategy will not be applicable. Additionally, the specificity of some reported aptamers are not satisfactory, such as the aptamer of adenosine triphosphate (ATP). In view of this, we investigated the interaction of the aptamer/GO complex with ATP and its analogs including adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine (AD). The result shows that the addition of ATP and its analogs respectively into the aptamer/GO complex gives rise to fluorescence recovery to different extents, following the order: ATP>AD>>DP> AMP. On the basis of the above finding, we proposed an effective approach to design robust GO-based biosensors for the assay of phosphatase and phosphorylase. Firstly, by employing the different binding ability of AD, AMP with GO/aptamer complex, and the dephosphorylation of AMP by alkaline phosphatase (ALP), the detection of ALP activity was realized using AMP as the substrate, and the detection limit reaches2.17U/L. Moreover, this design can be used to detect ALP activity in2%human serum. Based on the similar principle, using the difference in the affinity of GO/aptamer complex with ADP and ATP, and the phosphorylation of ADP by creatine kinase (CK) under certain conditions, the determination of CK activity was developed with a detection limit of0.73U/L. This design further broadens the detection range of GO-based DNA sensor. More importantly, the finding that the specificity of aptamer is improved after adsorbed on GO surfactant could be valuable for the screening system of aptamer, and would improve the specificity of aptamer.(4) After successful synthesis of a cationic conjugated polymer, poly [(9,9-bis (6'-N, N, N-trimethylammonium) hexyl)-fluorenylene phenylene dibromide](PFP), we investigated the interactions between PFP, GO and a fluorescein labeled single stranded DNA. We found that fluorescence resonance energy transfer (FRET) from PFP to probe DNA is inefficient when adding PFP into the probe DNA/GO complex. Based on this finding, a new method to reduce the high background signal of traditional PFP based DNA sensor was constructed by simply introducing GO into the system. Compared with the previously reported strategies such as the introduction of ethidium bromide and magnetic microparticles, this approach is simple, non-toxic and highly sensitive (detection limit:40pM), and would also promote the further application of GO and PFP in biosensors.